Nuclear/Cytosolic Fractionation Kit
Contents
1. Product Manual Nuclear Cytosolic Fractionation Kit Catalog Number AKR 172 100 preps FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research Introduction Cell Biolabs Nuclear Cytosolic Fractionation Kit provides a simple and fast tool to isolate nuclear extract from the cytoplasmic fraction of mammalian cells The procedure has been optimized to provide extraction with high protein recovery and low cross contamination in less than 2 hours The extracted protein fractions are functional and suitable for downstream assays such as DNA footprinting RNA splicing gel shift assays EMSA reporter assays enzyme activity assays and Western blotting Each kit provides sufficient quantities to perform 100 preps up to 5 x 10 cells each Related Products 1 AKR 102 Phospho Antibody Stripping Solution 2 AKR 103 PhosphoBlocker Blocking Reagent 1L 3 AKR 105 Phosphoprotein Purification Kit Kit Components Box 1 shipped at room temperature 1 Cytosol Extraction Buffer Hypotonic 10X Part No 217201 One bottle 10 mL 2 Cell Lysis Reagent Part No 217202 One bottle 5 mL of 10 Igepal CA 630 in 1X Cytosol Extraction Buffer CEB 3 Nuclear Extraction Buffer Part No 217203 One bottle 10 mL Box 2 shipped on blue ice packs 1 Dithiothreitol 1000X Part No 217204 One vial 100 uL of 1 M DTT 2 Protease Inhibitor2. Cocktail 100X Part No 217205 One vial 1 mL containing AEBSF Aprotinin Bestatin E64 Leupeptin and Pepstatin A in DMSO Materials Not Supplied 1 PBS 2 Microcentrifuge tubes 3 Microcentrifuge Storage Upon receiving aliquot and store Dithiothreitol and Protease Inhibitor Cocktail at 20 C and avoid multiple freeze thaw cycles Store all other components at 4 C Preparation of Reagents e 1X Cytosol Extraction Buffer CEB Dilute the 10X Cytosol Extraction Buffer to 1X with deionized water Stir to homogeneity N CELL BIOLABS INC r SK e Dithiothreitol Immediately before use dilute the Dithiothreitol 1 1000 with 1X Cytosol or Nuclear Extraction Buffer Stir to homogeneity Do not store diluted solutions e Protease Inhibitor Cocktail Immediately before use dilute the Protease Inhibitor Cocktail 1 100 with 1X Cytosol or Nuclear Extraction Buffer Stir to homogeneity Do not store diluted solutions Preparation of Samples I Adherent Cells 1 Au wWN Culture cells to approximately 80 90 confluence Aspirate the culture media and wash twice with PBS Detach the cells from the plates in PBS by scraping with a cell scraper Collect the solution into an appropriate conical centrifuge tube Centrifuge for 5 minutes 600 x g Discard the supernatant and immediately proceed to the Assay Protocol Section II Suspension Cells Or ee ee Collect the cells into an appropriate conical centrifuge tu
3. be Centrifuge for 5 minutes 600 x g Remove and discard the supernatant Wash the cells twice with PBS Centrifuge for 5 minutes at 600 x g Discard the supernatant and immediately proceed to the Assay Protocol Section Assay Protocol Important Note Perform the below steps at 2 8 C All buffers centrifuge rotors and equipment should be maintained at 2 8 C Before use Dithiothreitol and Protease Inhibitor Cocktail should be diluted according to the Preparation of Reagents section above I Cytosol Fractionation Protocol 1 Pi Collect cells up to 5 x 10 by centrifugation for 5 minutes at 4 C 600 x g 2 Wash the cells once with ice cold PBS 3 4 Remove and discard the supernatant Gently resuspend the cell pellet with 500 uL of ice cold 1X Cytosol Extraction Buffer containing DTT Protease Inhibitors by pipetting up and down Transfer the suspension into a prechilled microcentrifuge tube 6 Incubate on ice for 10 minutes 3 AN CELL BIOLABS INC N ZA 7 Add 25 uL of Cell Lysis Reagent and vortex for 10 seconds at the highest setting 8 Centrifuge for 10 minutes at 4 C 800 x g 9 Carefully transfer the supernatant cytoplasmic fraction to a clean chilled microcentrifuge tube The cytoplasmic fraction can be stored at 80 C for future use Note Make sure not to disturb remove the nuclei pellet 10 Gently resuspend the pellet with 500 uL of ice cold 1X Cytosol Extraction Buff
4. er containing DTT Protease Inhibitors by pipetting up and down Note This wash step is included to reduce cross contamination between fractions 11 Add 25 uL of Cell Lysis Reagent and vortex for 10 seconds at the highest setting 12 Centrifuge for 10 minutes at 4 C 800 x g 13 Carefully aspirate the supernatant and discard of this wash II Nuclear Protein Extraction Protocol 1 Gently resuspend the nuclear pellet with 100 uL of ice cold 1X Nuclear Extraction Buffer containing DTT Protease Inhibitors by pipetting up and down 2 Maintain on ice for 30 minutes vortexing for 10 seconds at the highest setting in 10 minute intervals 3 Centrifuge for 30 minutes at 4 C 14000 x g 4 Carefully transfer the supernatant nuclear protein extract to a clean chilled microcentrifuge tube The extract can be stored at 80 C for future use Note The nuclear extract typically yields protein concentrations of gt 1 mg mL If greater concentrations are desired resuspend the nuclear pellet in a smaller volume in step 1 above minimum of 25 uL II Other Considerations e For determining the protein content of extracts samples must be diluted 1 2 before running in the Bradford Protein Assay Buffer only controls must be performed concurrently DTT in the buffers is not compatible with the BCA Protein Assay e Nuclear Extraction Buffer is a high salt buffer containing 420 mM NaCl If salt removal is necessary dialysis or a desa
5. lting column may be used i CELL BIOLABS INC Example of Results The following figure demonstrates typical results seen with Cell Biolabs Nuclear Cytosolic Fractionation Kit One should use the data below for reference only w C N w C N 120 85 60 50 40 25 Anti a Tubulin Anti Lamin A C Figure 1 HEK293 Cell Fractionation Cytosolic and nuclear protein extracts were isolated from Human Embryonic Kidney 293 cells according to the Assay Protocol Whole cell W cytosol C and nuclear N fractions were immunoblotted with Anti a Tubulin left or Anti Lamin A C right at 1 ug mL Note Anti a Tubulin Calbiochem CP06 and Anti Lamin A C Sigma SAB4200236 are both mouse monoclonals Tubulin and Lamin are known to be cytosolic and nuclear specific proteins respectively Untreated Resuspended in Post CEB Hypotonic CEB Lysis Reagent Figure 2 HEK293 Trypan Blue Staining Human Embryonic Kidney 293 cells were stained with Trypan Blue at various steps during the fractionation protocol demonstrating complete lysis and high neuclei recovery Recent Product Citations 1 Nakamura S et al 2015 Novel roles for LIX1L in promoting cancer cell proliferation through ROS 1 mediated LIX1L phosphorylation Sci Rep doi 10 1038 srep 13474 g CELL BIOLABS INC A AA 2 Shinmura K et al 2015 NEIL1 p Gln282Stop variant is predominantly localized in the cytoplasm and exhibits reduced activit
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7. y in suppressing mutations Gene doi 10 1016 j gene 2015 06 043 3 Jeon Y J et al 2015 A set of NF kB regulated microRNAs induces acquired TRAIL resistance in lung cancer Proc Natl Acad Sci U S A 112 E3355 64 4 Ohtsuka S et al 2014 SQSTM1 p62 A170 regulates the severity of Legionella pneumophila pneumonia by modulating inflammasome activity Eur J Immunol 44 1084 1092 5 Zou J et al 2014 A TIR domain protein from E faecalis attenuates MyD88 mediated signaling and NF B activation PLoS One 9 e112010 Warranty These products are warranted to perform as described in their labeling and in Cell Biolabs literature when used in accordance with their instructions THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSED WARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE CELL BIOLABS sole obligation and purchaser s exclusive remedy for breach of this warranty shall be at the option of CELL BIOLABS to repair or replace the products In no event shall CELL BIOLABS be liable for any proximate incidental or consequential damages in connection with the products Contact Information Cell Biolabs Inc 7758 Arjons Drive San Diego CA 92126 Worldwide 1 858 271 6500 USA Toll Free 1 888 CBL 0505 E mail tech cellbiolabs com www cellbiolabs com 2012 2015 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in a
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